The long-term goal of this project is to understand how CD1d controls the immune regulatory function of Va14Ja18 NKT cells. In this application we propose to test the central hypothesis that the natural Va14Ja18 NKT cell antigen is a glycosphingolipid whose assembly with CD1d in MHC class II-enriched vesicles (CIIV/MIIC) requires lipid and protein chaperones. The rationale for this hypothesis is that CD1d controls the immune functions of NKT cells by presenting a putative self lipid antigen(s) whose chemical structure remains unknown. Currently, of all the lipid antigens known, only the synthetic anti-tumor agent alpha-galactosylceramide (alphaGalCer) activates almost all Va14Ja18 NKT cells. Current data suggest that CD1d assembles with invariant chain and/or phospholipids in the ER and exchanges these ligands for another cellular lipid in the CIIV/MIIC. Thence the antigen-loaded CD1d is presented to NKT cells. The chemical nature of the self lipid antigen(s) presented by CD1d to Va14Ja18 NKT cells and the mechanism(s) for biosynthetic assembly of CD1d1 as well as antigen loading in the CIIV/MIIC currently remain outstanding, unanswered questions of major immunological import. Our strategy to test the central hypothesis of this project will be to: a) Define the chemical structure of a natural Va14Ja18 NKT cell antigen to test the hypothesis that a natural antigen is a glycosphingolipid, b) Define the immune functions of a putative Va14Ja18 T cell ligand, thereby test the hypothesis that alpha-glucosylceramide is a natural antigen, c) Determine the mechanism(s) underlying the biosynthetic assembly of CD1 and the assembly of CD1d with antigen to test the hypothesis that this assembly requires lipid and protein chaperone(s), d) Define the molecular/structural features of the Va14Ja18 NKT cell receptor-antigen interface to test the hypothesis that co-operative antigen-TcR interaction, which permits sensitive antigen recognition, involves allosteric change at the ligand-receptor interface. Thus the goals of this proposal have the potential of providing fundamental new insights into our understanding of immune recognition, response and regulation. In keeping with our long-term goal, upon completion of this project we expect to elucidate the mechanism by which CD1d1 assembles in vivo and controls Va14Ja18 NKT cell function. Additionally, the identification of a natural antigen could lead to the development of natural therapeutics replacing alphaGalCer, which has been demonstrated to prevent the onset of several autoimmune diseases including type I diabetes and experimental autoimmune encephalomyelitis. Together, they will significantly advance our understanding of the physiological role of the CD1 antigen presentation system and NKT cells within the context of the immune system.